The proposed study is directed towards examining the influence of the membrane environment on the function and state of the nicotinic-cholinergic receptor. The approach will involve: (1) physical and kinetic studies on the purified membranes obtained from Torpedo electric organs, and (2) examining the topologic and pharmacologic properties of the receptor in culture where external prarameters can be varied to modulate receptor behavior. The studies with the purified membrane fragments will employ electron spin resonance using nitroxide analogues of cholinergic agonists, competition with alpha-toxin binding kinetics and irreversibly acting ligands to study agonist-induced transitions in receptor state. These transitions bear a close resemblance to receptor desensitization in situ. Studies of the influence of membrane structure and fluidity on the receptor state will employ fluorescence probes of membrane and alterations in membrane composition. To obtain membranes of the critical purity and uniformity necessary for these studies, we will rely on the affinity partitioning. We also plan to perfect this technique and ascertain whether it can be applied to systems containing receptors in lower density than those found in Torpedo. The cell culture studies will employ non-fusing muscle cell line, BC3H-1. This system will enable us to study receptor state, localization, pharmacologic specificity in relation to developmental parameters. Since this cell line also possesses beta-adrenergic receptors, we hope to study the topological relationships between the two receptor types. The high affinity antagonist 125 iodohydroxybenzylpindolol will be employed for localizing and characterizing the beta-receptor. Affinity partitioning should also prove useful for the cell culture receptors since it may allow us to separate membrane patches containing high and low densities of receptors. The BC3H-1 cell line and isolated membranes of high receptor density from Torpedo are most amenable to different experimental approaches, yet they should yield complementary information on the membrane associated receptor.